Donor organ shortages have led to hopes that xenotransplantation could serve as an alternative means of organ availability. Swine, particularly mini-swine, are an attractive alternative to non-human primate donors because of potentially greater availability, the reduced risk of zoonotic infections, appropriate size of organs and the reduced social and ethical concerns (Sachs, D. H. et al. 1976. Transplantation 22:559-567; Auchincloss, H. Jr. 1988. Transplantation 46:1-20). However, one of the major barriers to xenotransplantation is the phenomenon described as hyperacute rejection (Busch et al. 1972. Am. J. Pathology 79:31-57; Auchincloss, H. Jr. 1988. Transplantation 46:1-20). This phenomenon describes a very rapid and severe humoral rejection, which leads to destruction of the graft within minutes or hours of the transplant of the donor organ. Hyperacute rejection is apparently mediated by a complex series of events, including activation of the complement systems, activation of blood coagulation proteins, activation of endothelial cells and release of inflammatory proteins (Busch et al. 1972. Am. J. Pathology 79:31-57; Platt, J. L. 1992. ASAIO Journal 38:8-16). There is an accumulating body of information that implicates a group of pre-formed antibodies, the so-called natural antibodies, to be of fundamental importance in the hyperacute rejection seen in grafts between species. Species combinations in which the recipients of grafts have circulating antibodies that can initiate the hyperacute response to the donor species are described as discordant. Pigs and humans are one such discordant species combination.
The hyperacute rejection process is initiated when the natural antibodies of the recipient bind to cells of the donor organ (Platt et al. 1990. Transplantation 50:870-822; Platt et al. 1990. Immunology Today 11:450-456). It has been suggested that porcine N-linked carbohydrates carrying a terminal Galxcex11-3Galxcex21-4GlcNAc structure are the major targets for anti-swine xenoreactive human natural antibodies (Good et al. 1992. Transplantation Proceedings 24:559-562; Sandrin et al. 1993. Proc. Natl. Acad. Sci. USA 90:11391-11395). One major difference between the glycosylation pattern of swine tissues and human tissues is the presence of high levels of a terminal Galxcex11-3Galxcex21-4GlcNAc structure on swine cells and tissues. This structure is expressed at high levels in all lower mammals investigated, but is poorly expressed on cells and tissues of Old World monkeys, apes and humans (catarrhines) (Galili, U. and Swanson, K. 1992. Proc. Natl. Acad. Sci. USA 88:7401-7404; Galili et al. 1987. Proc. Natl. Acad. Sci. USA. 84:1369-1373). A specific transferase, UDP-Gal:Galxcex21xe2x86x924GlcNAc xcex11xe2x86x923-galactosyltransferase (EC 2.4.1.151; xcex1(1,3) galactosyltransferase) is responsible for the transfer of a terminal galactose to the terminal galactose residue of N-acetyllactosamine-type carbohydrate chains and lactosaminoglycans according to the reaction: 
where R may be a glycoprotein or a glycolipid (Blanken, W. M. and Van den Eijinden, D. H. 1985. J. Biol. Chem. 260:12927-12934). Thus the Galxcex11-3Galxcex21-4GlcNAc epitope. Full length CDNA sequences encoding the murine (Larsen et al. 1989. Proc. Natl. Acad. Sci. USA. 86:8227-8231) and bovine (Joziasse et al. 1989. J. Biol. Chem. 264:14290-14297) enzymes have been determined. In addition, the genomic organization of the murine a(1,3) galactosyltransferase gene has been established (Joziasse et al. 1992. J. Biol. Chem. 267:5534-5541). A partial sequence encoding the 3xe2x80x2 region of the porcine xcex1(1,3) galactosyltransferase cDNA gene has been determined (Dabkowski et al. 1993. Transplantation Proceedings. 25:2921) but the full length sequence has not been reported. The absence of the 5xe2x80x2 sequence is significant for the applications described herein. In contrast to the lower mammals, humans do not express the xcex1(1,3) galactosyltransferase. Furthermore, human sequences homologous to the murine sequence correspond to a processed pseudogene on chromosome 12 and an inactivated remnant on chromosome 9 (Shaper et al. 1992. Genomics 12:613-615).
In accordance with the invention, swine organs or tissues or cells that do not express xcex1(1, 3) galactosyltransferase will not produce carbohydrate moieties containing the distinctive terminal Galxcex11-3Galxcex21-4GlcNAc epitope that is a significant factor in xenogeneic, particularly human, transplant rejection of swine grafts. Further in accordance with the invention, is the aspect of diminishing the production of xcex1(1,3) galactosyltransferase to an extent sufficient to prevent the amount produced from providing carbohydrates with the Galxcex11-3Galxcex21-4GlcNAc epitope from being presented to the cell surface thereby rendering the transgenic animal, organ, tissue, cell or cell culture immunogenically tolerable to the intended recipient without requiring complete xcex1(1,3) galactosyltransferase gene suppression.
One principal aspect of the present invention is that the inventors have isolated the entire porcine xcex1(1,3) galactosyltransferase CDNA gene (SEQ. ID NO. 1). The identification, isolation and sequencing of the entire cDNA gene, now particularly providing the sequence of the 5xe2x80x2 end is an important advance because, as described in Example 2, this region has been identified as the most efficient for antisense targeting. Moreover, as compared with mouse and bovine homologous sequences (FIG. 2), this region of the xcex1(1,3) galactosyltransferase MRNA appears to deviate extensively between these species making it extremely unlikely that a use of xe2x80x9ccross-speciesxe2x80x9d antisense constructs would be successful.
Another principle aspect of this invention related to genetically altered animals, more specifically transgenic, chimeric or mosaic swine in which the expression of biologically active xcex1(1,3) galactosyltransferase is prevented in at least one organ, tissue or cell type. Transgenic animals carry a gene which has been introduced into the germline of the animal, or an ancestor of the animal, at an early developmental stage. The genetic alteration in transgenic animals is stably incorporated into the genome as a result of intentional experimental intervention. Typically, this results from the addition of exogenous foreign DNA or novel constructs (Palmiter et al. 1986. Ann. Rev. Genet. 20:465). With the advent of embryonic stem (ES) cells and specific gene targeting, the definition of transgenesis now includes specific modification of endogenous gene sequences by direct experimental manipulation and by stable incorporation of DNA that codes for effector molecules that modulate the expression of endogenous genes (Gossler et al. 1986. Proc. Natl. Acad. Sci. USA. 83:9065; Schwarzberg et al. 1989. Science 246:799; Joyner et al. 1989. Nature 338:153).
One preferred approach for generating a transgenic animal involves micro-injection of naked DNA into a cell, preferentially into a pronucleus of an animal at an early embryonic stage (usually the zygote/one-cell stage). DNA injected as described integrates into the native genetic material of the embryo, and will faithfully be replicated together with the chromosomal DNA of the host organism. This allows the transgene to be passed to all cells of the developing organism including the germ line. Transgene DNA that is transmitted to the germ line gives rise to transgenic offspring. If transmitted in a Mendelian fashion, half of the offspring will be transgenic. All transgenic animals derived from one founder animal are referred to as a transgenic line. If the injected transgene DNA integrates into chromosomal DNA at a stage later than the one cell embryo not all cells of the organism will be transgenic, and the animal is referred to as being genetically mosaic. Genetically mosaic animals can be either germ line transmitters or non-transmitters. The general approach of microinjection of heterologous DNA constructs into early embryonic cells is usually restricted to the generation of dominant effects, i.e., one allele of the transgene (hemizygous) causes expression of a phenotype (Palmiter et al. 1986. Ann. Rev. Genetics 20:465.)
In another preferred approach, animals are genetically altered by embryonic stem (ES) cell-mediated transgenesis (Gossler et al. 1986, Proc. Natl. Acad. Sci. USA. 83:9065). ES cell lines are derived from early embryos, either from the inner cell mass (ICM) of a blastocyst (an embryo at a relatively early stage of development) or migrating primordial germ cells (PGC) in the embryonic gonads. They have the potential to be cultured in vitro over many passages (i.e. are conditionally immortalized), and they are pluripotent, or totipotent (i.e. are capable of differentiating and giving rise to all cell types. ES cells can be introduced into a recipient blastocyst which is transferred to the uterus of a foster mother for development to term. A recipient blastocyst injected with ES cells can develop into a chimeric animal, due to the contributions from the host embryo and the embryonic stem cells. ES cells can be transfected with heterologous gene constructions that may cause either dominant effects, inactivate whole genes or introduce subtle changes including point mutations. Subsequent to clonal selection for defined genetic changes, a small number of ES cells can be reintroduced into recipient embryos (blastocysts or morulae) where they potentially differentiate into all tissues of the animal including the germ line and thus, give rise to stable lines of animals with designed genetic modifications. Totipotent porcine embryonic stem cells can be genetically altered to have a heterozygous (+/xe2x88x92) mutant, preferably null mutant allele, particularly one produced by homologous recombination in such embryonic stem cells. Alternatively, gene targeting events by homologous recombination can be carried out at the same locus in two consecutive rounds yielding clones of cells that result in a homozygous (xe2x88x92/xe2x88x92) mutant, preferably a null mutant (Ramirez-Solis et al. 1993. Methods in Enzymol. 225:855).
In one preferred embodiment of this invention a DNA sequence is integrated into the native genetic material of the swine and produces antisense RNA that binds to and prevents the translation of the native MRNA encoding xcex1(1,3) galactosyltransferase in the transgenic swine.
In a particularly preferred embodiment the genome of the transgenic swine is modified to include a construct comprising a DNA complementary to that portion of the xcex1(1,3) galactosyltransferase coding region that will prevent expression of all or part of the biologically active enzyme. As the term is used xe2x80x9cintegrated antisense sequencexe2x80x9d means a non-native nucleic acid sequence integrated into the genetic material of a cell that is transcribed (constitutively or inducibly) to produce an MRNA that is complementary to and capable of binding with an MRNA produced by the genetic material of the cell so as to regulate or inhibit the expression thereof.
In another embodiment of the invention, cells or cell lines from non-mutant swine are made with the xcex1(1,3) galactosyltransferase inactivated on one or both alleles through the use of an integrated antisense sequence which binds to and prevents the translation of the native MRNA encoding the xcex1(1,3) galactosyltransferase in said cells or cell lines. The integrated antisense sequence, such as the RNA sequence transcribed in Example 3 is delivered to the cells by various means such as electroporation, retroviral transduction or lipofection.
In another preferred embodiment, the transgenic swine is made to produce a ribozyme (catalytic RNA) that cleaves the xcex1(1,3) galactosyltransferase mRNA with specificity. Ribozymes are specific domains of RNA which have enzymatic activity, either acting as an enzyme on other RNA molecules or acting intramolecularly in reactions such as self-splicing or self-cleaving (Long, D. M. and Uhlenbeck, O. C. 1993. FASEB Journal. 7:25-30). Certain ribozymes contain a small structural domain generally of only about 30 nucleotides called a xe2x80x9chammerheadxe2x80x9d. The hammerhead is a loop of RNA that is flanked by two linear domains that are specific complements to domains on the substrate to be cleaved. The site on the hammerhead ribozyme that effects the cleavage of substrate is the base of the stem loop or hammerhead. As shown in FIG. 3, the ribozymes of the present invention have flanking sequences complementary to domains near the 5xe2x80x2end of the xcex1(1,3) galactosyltransferase cDNA gene.
The DNA for the ribozymes is integrated into the genetic material of an animal, tissue or cell and is transcribed (constitutively or inducibly) to produce a ribozyme which is capable of selectively binding with and cleaving the xcex1(1,3) galactosyltransferase mRNA. As it is a catalytic molecule, each such ribozyme is capable of cleaving multiple substrate molecules.
The catalytic xe2x80x9cstem loopxe2x80x9d of the ribozyme is flanked by sequences complementary to regions of the xcex1(1,3) galactosyltransferase MRNA. In a particularly preferred embodiment the transgenic swine is modified to integrate a construct comprising the DNA coding for that portion of catalytic RNA necessary to inactivate the mRNA of the xcex1(1,3) galactosyltransferase operably linked to a promoter therefor.
In another embodiment of the invention, cells or cell lines from non-mutant swine are made with the xcex1(1,3) galactosyltransferase inactivated on one or both alleles through the use of an integrated ribozyme sequence which binds to and cleaves the native mRNA encoding the xcex1(1,3) galactosyltransferase in said cells or cell lines. The integrated ribozyme sequence, such as the RNA sequence transcribed in Example 4 is delivered to the cells by various means such as electroporation, retroviral transduction or lipofection.
In another preferred embodiment, using cultured porcine embryonic stem cells, a mutation, preferably a null mutation is introduced by gene targeting at the native genomic locus encoding xcex1(1,3) galactosyltransferase. Gene targeting by homologous recombination in ES cells is performed using constructs containing extensive sequence homology to the native gene, but specific mutations at positions in the gene which are critical for generating a biologically active protein. Therefore, mutations can be located in regions important for either translation, transcription or those coding for functional domains of the protein. Selection for ES clones that have homologously recombined a gene targeting construct, also termed gene xe2x80x9cknock outxe2x80x9d construct, can be achieved using specific marker genes. The standard procedure is to use a combination of two drug selectable markers including one for positive selection (survival in the presence of drug, if marker is expressed) and one for negative selection (killing in the presence of the drug, if marker is expressed) (Mansour et al., 1988. Nature 336:348) One preferred type of targeting vector includes the neomycin phosphotransferase (neo) gene for positive selection in the drug G418, as well as the Herpes Simplex Virus-thymidine kinase (HSV-tk) gene for selective killing in gancyclovir. Drug selection in G418 and gancyclovir, also termed positive negative selection (PNS) (Mansour et al. 1988. Nature 336:348; Tubulewicz et al. 1991. Cell 65:1153) allows for enrichment of ES cell clones that have undergone gene targeting, rather than random integration events. Confirmation of homologous recombination events is performed using Southern analysis.
The design of the xcex1(1,3) galactosyltransferase targeting construct is described in Example 6. The procedure as applied here uses a positive selection (survival) based on integration of the neo (neomycin resistance), preferably in inverse orientation to the endogenous xcex1(1,3) galactosyltransferase gene locus in a cassette with the phosphoglycerate kinase (PGK-1) promoter and with flanking oligonucleotides complementary to two separate regions of the xcex1(1,3) galactosyltransferase gene sequence. It is understood that other positive selectable markers may be used instead of neo. The neo gene is linked with its promoter to be under control thereof. Downstream from the second flanking sequence is the HSV-tk gene which, if integrated into the genome encodes for production of thymidine kinase making the cell susceptible to killing by gancyclovir (negative selection). The integration of the neo gene but not the HSV-tk gene occurs only where integration into the xcex1(1,3) galactosyltransferase gene has occurred and provides for both positive and negative selection of the cells so transformed.
In another preferred embodiment, using isogenic DNA, it has become possible to achieve high frequency homologous recombination even in biological systems, such as zygotes, which do not lend themselves to the use of elaborate selection protocols and were, therefore, previously not suitable candidates for the isolation of cells which showed positive marker attributes indicating homologous recombination. This use of targeting vectors which include isogenic DNA which is substantially identical to that of chromosomal segments of the target recipient cell, can be used to target zygotes which thereafter develop into transgenic animals. The use of these zygote cells is preferably to produce a mutation, preferably a null mutation, at the chromosomal locus encoding xcex1 (1,3) galactosyltransferese. Thus, these vectors contain extensive sequence homology to the native gene, but also contain specific mutations at segments in the gene which are critical for generating a biologically active protein. Therefore, mutations can be located in regions important for either translation, transcription, or those coating for functional domains of the protein. The high percentage of homologous recombination achieved using isogenic DNA makes it possible to avoid the need for selection of clones that have homologously recombined the gene targeting construct, as described above with respect to the xe2x80x9cknock outxe2x80x9d embodiment thereby making it possible to avoid the need for the standard selection procedure described above.
More particularly, in this embodiment, PCR is used to identify and extract 1-2 kb DNA fragments, which are then subjected to restriction fragment length polymorphism digestions to identify areas or alleles that are most abundantly present in the line of mini-swine selected for zygote injection. Known insertion vectors are used, however, replacement vectors also described in the art could also be used. It is also possible to use DNA sequences with an isogenic replacement vector that require only a few kilobases of uninterrupted isogenic DNA. As such, it is possible to effect highly efficient homologous recombination such that it is not necessary to screen targeting vector recombined zygotes. Rather, genomic DNA screening using PCR can be done after the piglets are born. These transgenic founder animals, which are observed to have the transgene targeted to the native xcex1 (1,3) galactosyltransferese locus (i.e. that are heterozygous null mutant for that gene, are grown and interbred to produce animals that are homozygous null mutant for this locus, resulting in the knock out of the xcex1 (1,3) galactosyltransferese gene which can be confirmed by antibody or lectin binding assays.
The swine is preferably an xcex1(1,3) galactosyltransferase negative swine grown from a porcine oocyte whose pronuclear material has been removed and into which has been introduced a totipotent porcine embryonic stem cell using protocols for nuclear transfer (Prather et al. 1989, Biol. Reprod. 41:414) ES cells used for nuclear transfer are negative for the expression of xcex1(1,3) galactosyl transferase, or alternatively, totipotent ES cells used for nuclear transfer are mutated in a targeted fashion in at least one allele of the xcex1(1,3) galactosyltransferase gene.
The swine is preferably lacking expression of the xcex1(1,3) galactosyltransferase gene and bred from chimeric animals which were generated from ES cells by blastocyst injection or morula aggregation. ES cells used to generate the preferably null-mutated chimeric animal were mutated at least in one allele of the xcex1(1,3) galactosyltransferase gene locus, using gene targeting by homologous recombination.
A chimeric swine is preferably constituted by ES cells mutated in one allele of the xcex1(1,3) galactosyltransferase gene. Derived from mutated ES cells are also germ cells, male or female gametes that allow the mutation to be passed to offspring, and allow for breeding of heterozygous mutant sibling pigs to yield animals homozygous mutant at the xcex1(1,3) galactosyltransferase locus. Also described is a swine, deficient for an xcex1(1,3) galactosyltransferase protein (i.e., characterized by lack of expression of xcex1(1,3) galactosyltransferase protein) and have little, if any, functional Galxcex11-3Galxcex21-4GlcNAc epitope-containing carbohydrate antigen on the cell surface are produced. Further described are methods of producing transgenic swine and methods of producing tissue from heterozygous swine or homozygous swine of the present invention. The present invention also relates to cell lines, such as swine cell lines, in which the xcex1(1,3) galactosyltransferase gene is inactivated on one or both alleles and use of such cell lines as a source of tissue and cells for transplantation.
Tissues, organs and purified or substantially pure cells obtained from transgenic swine, more specifically from hemizygous, heterozygous or homozygous mutant animals of the present invention can be used for xenogeneic transplantation into other mammals including humans in which tissues, organs or cells are needed. The xcex1(1,3) galactosyltransferase inactive cells can themselves be the treatment or therapeutic/clinical product. For example, keratinocytes rendered xcex1(1,3) galactosyltransferase inactive can be used for macular degeneration and pancreatic cells rendered xcex1(1,3) galactosyltransferase deficient can be used to replace or restore pancreatic products and functions to a recipient. In another embodiment, xcex1(1,3) galactosyltransferase inactive cells produced by the present method are further manipulated, using known methods, to introduce a gene or genes of interest, which encode(s) a product(s), such as a therapeutic product, to be provided to a recipient. In this embodiment, the xcex1(1,3) galactosyltransferase deficient tissue, organ or cells serve as a delivery vehicle for the encoded product (s). For example, xcex1(1,3) galactosyltransferase deficient cells, such as fibroblasts or endothelial cells, can be transfected with a gene encoding a therapeutic product, such as cytokines that augment donor tissue engraftment, Factor VIII, Factor IX, erythropoietin, insulin, human major histocompatibility (MHC) molecules or growth hormone, and introduced into an individual in need of the encoded product.
Alternatively, recipient blastocysts are injected or morulae are aggregated with totipotent embryonic stem cells yielding chimeric swine containing at least one allele of a mutated, preferably null-mutated xcex1(1,3) galactosyltransferase gene produced by homologous recombination. A chimeric swine is preferably constituted by ES cells mutated in one allele of the xcex1(1,3) galactosyltransferase gene. Derived from mutated ES cells are also germ cells that allow the mutation to be passed to offspring, and breeding of heterozygous mutant sibling pigs to yield animals homozygous mutant at the xcex1(1,3) galactosyltransferase locus. Also described is a swine, deficient for an xcex1(1,3) galactosyltransferase protein (i.e., characterized by essentially no expression of xcex1(1,3) galactosyltransferase protein) and with little, if any, functional Galxcex11-3Galxcex21-4GlcNAc epitope-containing carbohydrate antigen on the cell surface are produced. Further described are methods of producing transgenic swine and methods of producing tissue from heterozygous swine or homozygous swine of the present invention. The present invention also related to cell lines, such as swine cell lines, in which the xcex1(1,3) galactosyltransferase gene is inactivated on one or both alleles and use of such cell lines as a source of tissue, organs and cells for transplantation.